Constructing Replicons and Reporter Plasmids

The detailed construction of R1 replicon has been described previously (Su et al., 2020 (link)). Briefly, for R1 3D^D330A, in which Asp330 was mutated to an Ala to disrupt the enzymatic activity, was constructed by PCR-based site-directed mutagenesis using the R1 replicon as a template (Su et al., 2020 (link)). The monocistronic reporter plasmid (IRES-Luc), which contains EV-A71 IRES at the 5′end and drives the translation of the luciferase protein, was described previously (Su et al., 2018 (link)). To construct pFlag-CMV2-HSPA6, HSPA6 gene was amplified from the cDNA of EV-A71 infected RD cells by nested PCR and then cloned into pFlag-CMV2 (Sigma-Aldrich) at NotI and KpnI sites to generate pFlag-HSPA6. A wobble HSPA6 mutation (nucleotides 7–9 GCC→GCA), which resists the action of sgRNA used in knockout cells, was generated by site-direct mutagenesis. HCV IRES-Luc was previously described (Su et al., 2018 (link)). CV-A16 IRES-Luc and Echo 9 IRES-Luc were gifts from Dr. Szu-Hao Kung (National Yang-Ming University, Taiwan) (Hou et al., 2016 (link)). EMCV IRES-Luc was generated by inserting firefly Luc gene into the pTM1 plasmid (Addgene).